Effect of probe characteristics on the subtractive hybridization efficiency of human genomic DNA

<p>Abstract</p> <p>Background</p> <p>The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics.</p> <p>Findings</p> <p>In this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA.</p> <p>Conclusions</p> <p>The characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.</p

Comparative clinical and transcriptomal profiles of breast cancer between French and South Mediterranean patients show minor but significative biological differences.

Erratum in :Cancer Genomics Proteomics. 2008 Nov-Dec;5(6):353.Cancer Genomics Proteomics. 2009 Jan-Feb;6(1):73.International audienceBACKGROUND: In Western countries, breast cancer incidence and mortality are higher than in Mediterranean countries. These differences have been ascribed to environmental factors but also to late-stage diagnostic and biological specific characteristics.PATIENTS AND METHODS: Between September 2002 and September 2005, we collected clinical data by phone counselling 180 French and Mediterranean breast cancer patients and performed microarray experiments.RESULTS: Characteristics of breast cancer in patients from Lebanon, Tunisia and Morocco were more aggressive (more SBR grade III and positive node invasion) and patients were 10 years younger at diagnosis. Sixteen differentially expressed genes such as MMP9, VEGF, PHB1, BRCA1, TFAP2C, GJA1 and TFF1 were also found. Additionally, an up-regulation of cytokeratins KRT8 and KRT18 may indicate a luminal B subtype in "South" (Lebanon, Tunisia and Morocco) tumors while "North" (France) tumors may more frequently be luminal A type.CONCLUSION: This study allowed the identification of specific clinical and transcriptomic parameters in patients from South Mediterranean countries

Site-Specific Immobilization of DNA in Glass Microchannels via Photolithography

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